AlanFung:Protocol/Plasmid DNA amplification

From ZhangLabWiki
Revision as of 21:58, 20 September 2010 by >Alan6017518 (→‎Grow cells)
Jump to navigation Jump to search

Plasmid DNA Amplification

For pRFP-C-RS based HuSH vector from Origene

Dilution of Plasmid solution

  • Add 50ul of dH2O into each tube
  • Vortex the tubes to resuspend the DNA
  • Pipette 1ul of this soultion to another tube
  • Add 99ul H2O Concentration of the DNA solution should be 1ng/ul
  • Notes: Store plasmid solution at -20C

Grow cells on LB-Plates

  • Thaw transformation competent E.Coli cells on ice
  • Perform transformation with 1-2ul of diluted shRNA plasmid
  • Plate out the transformants on LB-Chloramphenicol plates
  • Incubate overnight at 37C, until colonies appear

Grow cells in LB-Broth

  • The following day inoculate single bacterial colonies into 5ml of LB-KChloramphenicol and grow them overnight

Purify DNA plasmids

Purify DNA plasmids form the culture using a miniprep DNA isolation kit

Prepare plasmids for packaging

  • Resuspend the DNA in 50ul of TE solution
  • Determine the concentration of the samples and make sure it meets the core requirements